Structured Review

GeneTex β tubulin
A3A and A3B were contained in the mitochondria. ( a ) W12 cells were transfected with an expression vector coding A3A or HA-A3B, and cultivated in maintenance medium for 48 h. Their lysates were fractionated into cytosolic and mitochondrial fractions, and subjected to western blotting analysis. ( b , c ) W12 cells were induced to differentiate under high extracellular calcium concentration, and fractionated into cytosol and mitochondrial fraction, ( b ) Each fraction was subjected to immunoblotting of A3s, HSP60 (a mitochondrial marker), and <t>β-tubulin.</t> ( c ) DNA was extracted from each fraction and subjected to 3D-PCR analysis targeting COI .
β Tubulin, supplied by GeneTex, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Keratinocyte differentiation induces APOBEC3A, 3B, and mitochondrial DNA hypermutation"

Article Title: Keratinocyte differentiation induces APOBEC3A, 3B, and mitochondrial DNA hypermutation

Journal: Scientific Reports

doi: 10.1038/s41598-018-27930-z

A3A and A3B were contained in the mitochondria. ( a ) W12 cells were transfected with an expression vector coding A3A or HA-A3B, and cultivated in maintenance medium for 48 h. Their lysates were fractionated into cytosolic and mitochondrial fractions, and subjected to western blotting analysis. ( b , c ) W12 cells were induced to differentiate under high extracellular calcium concentration, and fractionated into cytosol and mitochondrial fraction, ( b ) Each fraction was subjected to immunoblotting of A3s, HSP60 (a mitochondrial marker), and β-tubulin. ( c ) DNA was extracted from each fraction and subjected to 3D-PCR analysis targeting COI .
Figure Legend Snippet: A3A and A3B were contained in the mitochondria. ( a ) W12 cells were transfected with an expression vector coding A3A or HA-A3B, and cultivated in maintenance medium for 48 h. Their lysates were fractionated into cytosolic and mitochondrial fractions, and subjected to western blotting analysis. ( b , c ) W12 cells were induced to differentiate under high extracellular calcium concentration, and fractionated into cytosol and mitochondrial fraction, ( b ) Each fraction was subjected to immunoblotting of A3s, HSP60 (a mitochondrial marker), and β-tubulin. ( c ) DNA was extracted from each fraction and subjected to 3D-PCR analysis targeting COI .

Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Concentration Assay, Marker, Polymerase Chain Reaction

2) Product Images from "Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome"

Article Title: Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome

Journal: Orphanet Journal of Rare Diseases

doi: 10.1186/s13023-015-0235-8

Characterization of the bone and osteoblast pathology. A . Photograph of the bone biopsy. B . Steady state SMS mRNA levels relative to GAPDH expression in cultured fibroblasts and osteoblasts. The patient’s cells did not differ significantly from controls. Data were derived by qRT-PCR analysis of 3 independent extractions of total RNA. C . Immunoblot showing steady state SMS protein expression in patient and control osteoblasts. ß-tubulin is shown as a loading control. D . Graph showing steady state SMS protein levels in the patient and control hBMSCs relative to ß-tubulin levels; there was no significant difference. The data are based on 3 independent experiments for each cell line. E - J . Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E-G) and Patient II-1 (H-J) hBMSCs. SMS protein is shown in red and the nucleus is shown in blue. K . Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control hBMSCs. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. L . Polyamine quantification in fibroblasts and osteoblasts. Note that the patient hBMSCs have a more striking imbalance of spermidine and spermine levels than do the patient fibroblasts, * p
Figure Legend Snippet: Characterization of the bone and osteoblast pathology. A . Photograph of the bone biopsy. B . Steady state SMS mRNA levels relative to GAPDH expression in cultured fibroblasts and osteoblasts. The patient’s cells did not differ significantly from controls. Data were derived by qRT-PCR analysis of 3 independent extractions of total RNA. C . Immunoblot showing steady state SMS protein expression in patient and control osteoblasts. ß-tubulin is shown as a loading control. D . Graph showing steady state SMS protein levels in the patient and control hBMSCs relative to ß-tubulin levels; there was no significant difference. The data are based on 3 independent experiments for each cell line. E - J . Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E-G) and Patient II-1 (H-J) hBMSCs. SMS protein is shown in red and the nucleus is shown in blue. K . Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control hBMSCs. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. L . Polyamine quantification in fibroblasts and osteoblasts. Note that the patient hBMSCs have a more striking imbalance of spermidine and spermine levels than do the patient fibroblasts, * p

Techniques Used: Expressing, Cell Culture, Derivative Assay, Quantitative RT-PCR

Segregation, mutational analysis, and functional consequences of a novel SMS variant. A . Pedigree of the family of the propositi. Affected males are shown by black squares. B . Sanger sequencing chromatograms showing the segregation of the SMS mutation NM_004595.4:c.443A > G from the carrier mother to the affected boys. The unaffected father did not have this mutation. C . Conservation of the p.Gln148 (p.Q148) residue across species. D . Drawing of the human SMS protein crystal complexed with spermidine and 5-methylthioadenosine. The mutated amino acid (Gln148) is highlighted in yellow [Mac PyMOL [ 23 ]]. E - J . Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E, F) , Patient II-1 (G, H) and Patient II-3 (I, J) skin fibroblasts. SMS protein is shown in red and the nucleus is shown in blue. K . Immunoblot of skin fibroblast lysates showing reduced SMS protein levels in the patients (II-1, II-3) compared to an unaffected control (cnt). Tubulin is shown as a loading control. L . Graph showing steady state SMS protein levels in the patient and control fibroblasts relative to ß-tubulin levels. The data are based on 3 independent experiments for each cell line. M . Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control fibroblasts. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. The data are based on 2 independent experiments for each cell line. N . SMS enzyme activity (spermidine d8 peak per hour) in lymphoblasts of unaffected individuals (Cnt), a cohort of 4 individuals with SRS (SRS) and patient II-1, * p
Figure Legend Snippet: Segregation, mutational analysis, and functional consequences of a novel SMS variant. A . Pedigree of the family of the propositi. Affected males are shown by black squares. B . Sanger sequencing chromatograms showing the segregation of the SMS mutation NM_004595.4:c.443A > G from the carrier mother to the affected boys. The unaffected father did not have this mutation. C . Conservation of the p.Gln148 (p.Q148) residue across species. D . Drawing of the human SMS protein crystal complexed with spermidine and 5-methylthioadenosine. The mutated amino acid (Gln148) is highlighted in yellow [Mac PyMOL [ 23 ]]. E - J . Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E, F) , Patient II-1 (G, H) and Patient II-3 (I, J) skin fibroblasts. SMS protein is shown in red and the nucleus is shown in blue. K . Immunoblot of skin fibroblast lysates showing reduced SMS protein levels in the patients (II-1, II-3) compared to an unaffected control (cnt). Tubulin is shown as a loading control. L . Graph showing steady state SMS protein levels in the patient and control fibroblasts relative to ß-tubulin levels. The data are based on 3 independent experiments for each cell line. M . Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control fibroblasts. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. The data are based on 2 independent experiments for each cell line. N . SMS enzyme activity (spermidine d8 peak per hour) in lymphoblasts of unaffected individuals (Cnt), a cohort of 4 individuals with SRS (SRS) and patient II-1, * p

Techniques Used: Functional Assay, Variant Assay, Sequencing, Mutagenesis, Expressing, Activity Assay

3) Product Images from "Loss of HSulf-1: The Missing Link between Autophagy and Lipid Droplets in Ovarian Cancer"

Article Title: Loss of HSulf-1: The Missing Link between Autophagy and Lipid Droplets in Ovarian Cancer

Journal: Scientific Reports

doi: 10.1038/srep41977

HSulf-1 loss promotes lipid droplet biogenesis and defective autophagy in OV202, TOV2223 and HSulf-1 knockout mouse embryonic fibroblast cells. ( A ) Protein expression of HSulf-1 was assessed by western blot in OV202 clones (NTC, Sh1, Sh2 and Cl7 cells). β-Actin was probed as a loading control. ( B ) OV202NTC, Sh1, Sh2 and Cl7 cells were labeled with Bodipy 493/503 (green) and DAPI (Blue) to determine the cytoplasmic lipid droplets (LD) and nuclei respectively. ( C ) Mean fluorescence of Bodipy in NTC, Sh1, Sh2 and Cl7 is measured using Carl Zeiss Zen (2009) software and the data is plotted as a bar diagram. Sh1 and Sh2 cells were compared to NTC cells whereas Cl7 cells were compared to Sh1 cells. In both the cases, (p ≤ 0.05) ( D ) Representative trans-electron microscopic (TEM) images of NTC, Sh1, Sh2 and Cl7 cells are shown; cytoplasmic LDs are marked with green arrows, whereas red arrows indicate autophagic vesicles (AV). ( E and F ) LDs and AVs quantified in NTC, Sh1 and Sh2 cells and plotted in a bar graph. Sh1 and Sh2 cells were compared to NTC cells and, Cl7 cells were compared to Sh1 cells. In all the cases p ≤ .0.01 ( G ) Protein expression of HSulf-1 in TOV2223 NTC and Sh1 cells is detected by immunoblot analysis; β-actin was probed as a loading control. ( H ) Bodipy staining performed in TOV2223 NTC and Sh1 cells to detect LDs. ( I ). Representative TEM images of TOV2223 NTC and Sh1 cells are shown. ( J ) AVs and LDs in TOV2223 cells are quantified. We compared TOV2223 NTC cells to TOV2223 HSulf-1 knockdown Sh cells and found significant differences in the # of AVs and LDs ( K ) Protein expression of HSulf-1 in Mouse Embryonic Fibroblast (MEF) wild type (WT) and HSulf-1 knock out (Sulf-1 −/− ) cells is detected by western blotting. β-Tubulin was used as a loading control. ( L ) MEF cells were labeled with Bodipy 493/503 (green) to determine cytoplasmic LDs. Data are shown as mean ± SD of 3 replicates per treatment. *P
Figure Legend Snippet: HSulf-1 loss promotes lipid droplet biogenesis and defective autophagy in OV202, TOV2223 and HSulf-1 knockout mouse embryonic fibroblast cells. ( A ) Protein expression of HSulf-1 was assessed by western blot in OV202 clones (NTC, Sh1, Sh2 and Cl7 cells). β-Actin was probed as a loading control. ( B ) OV202NTC, Sh1, Sh2 and Cl7 cells were labeled with Bodipy 493/503 (green) and DAPI (Blue) to determine the cytoplasmic lipid droplets (LD) and nuclei respectively. ( C ) Mean fluorescence of Bodipy in NTC, Sh1, Sh2 and Cl7 is measured using Carl Zeiss Zen (2009) software and the data is plotted as a bar diagram. Sh1 and Sh2 cells were compared to NTC cells whereas Cl7 cells were compared to Sh1 cells. In both the cases, (p ≤ 0.05) ( D ) Representative trans-electron microscopic (TEM) images of NTC, Sh1, Sh2 and Cl7 cells are shown; cytoplasmic LDs are marked with green arrows, whereas red arrows indicate autophagic vesicles (AV). ( E and F ) LDs and AVs quantified in NTC, Sh1 and Sh2 cells and plotted in a bar graph. Sh1 and Sh2 cells were compared to NTC cells and, Cl7 cells were compared to Sh1 cells. In all the cases p ≤ .0.01 ( G ) Protein expression of HSulf-1 in TOV2223 NTC and Sh1 cells is detected by immunoblot analysis; β-actin was probed as a loading control. ( H ) Bodipy staining performed in TOV2223 NTC and Sh1 cells to detect LDs. ( I ). Representative TEM images of TOV2223 NTC and Sh1 cells are shown. ( J ) AVs and LDs in TOV2223 cells are quantified. We compared TOV2223 NTC cells to TOV2223 HSulf-1 knockdown Sh cells and found significant differences in the # of AVs and LDs ( K ) Protein expression of HSulf-1 in Mouse Embryonic Fibroblast (MEF) wild type (WT) and HSulf-1 knock out (Sulf-1 −/− ) cells is detected by western blotting. β-Tubulin was used as a loading control. ( L ) MEF cells were labeled with Bodipy 493/503 (green) to determine cytoplasmic LDs. Data are shown as mean ± SD of 3 replicates per treatment. *P

Techniques Used: Knock-Out, Expressing, Western Blot, Clone Assay, Labeling, Fluorescence, Software, Transmission Electron Microscopy, Staining

4) Product Images from "Development of a potent Zika virus vaccine using self-amplifying messenger RNA"

Article Title: Development of a potent Zika virus vaccine using self-amplifying messenger RNA

Journal: Science Advances

doi: 10.1126/sciadv.aba5068

In vitro characterization of ZIKV SAM constructs. ( A ) Potency of the ZIKV SAM constructs. The RNA was in vitro transcribed, and 0.1 μg of the RNA from each vaccine construct was electroporated into BHK cells. Cells were collected 24 hours later and stained with an anti-dsRNA antibody. The upper and lower panels show the percentage and MFI of dsRNA-positive cells determined using flow cytometry, respectively. Error bars represent the SD of technical triplicates. Shown are the representatives of at least two experiments. ( B ) ZIKV SAM RNA expression of prM-E protein. BHK cells were electroporated with 4.0 μg of the indicated SAM RNA. Twenty-four hours later, cells were collected and lysates were analyzed by immunoblotting using antibody 4G2 specific to flavivirus E protein or to β-tubulin. Shown are the representatives of at least two experiments.
Figure Legend Snippet: In vitro characterization of ZIKV SAM constructs. ( A ) Potency of the ZIKV SAM constructs. The RNA was in vitro transcribed, and 0.1 μg of the RNA from each vaccine construct was electroporated into BHK cells. Cells were collected 24 hours later and stained with an anti-dsRNA antibody. The upper and lower panels show the percentage and MFI of dsRNA-positive cells determined using flow cytometry, respectively. Error bars represent the SD of technical triplicates. Shown are the representatives of at least two experiments. ( B ) ZIKV SAM RNA expression of prM-E protein. BHK cells were electroporated with 4.0 μg of the indicated SAM RNA. Twenty-four hours later, cells were collected and lysates were analyzed by immunoblotting using antibody 4G2 specific to flavivirus E protein or to β-tubulin. Shown are the representatives of at least two experiments.

Techniques Used: In Vitro, Construct, Staining, Flow Cytometry, RNA Expression

5) Product Images from "Osteoblast-secreted factors mediate dormancy of metastatic prostate cancer in the bone via activation of the TGFβRIII-p38MAPK-pS249/T252RB pathway"

Article Title: Osteoblast-secreted factors mediate dormancy of metastatic prostate cancer in the bone via activation of the TGFβRIII-p38MAPK-pS249/T252RB pathway

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-17-1051

Dominant-negative p38MAPK prevents dormancy induction by TGFβ2 or GDF10 in PCa cells in vitro (A) C4-2B4 cells stably-expressing empty vector or p38DN were examined by western blot for p-p38 followed by p38MAPK. β-tubulin, loading control. (B) Cells in (A) were treated with 50 ng/ml TGFβ2 or GDF10 for 60 min and immunostained for p-p38. (C) Cells in (A) were treated with TGFβ2 or GDF10 for 72 h. Cellular quiescence was determined by live-cell imaging. n cells were monitored in multiple experiments: C4-2B4-Vector (N=5–7), C4-2B4-p38DN (N=4–5). P values are by t test. (D) C4-2b-Vector or C4-2b-p38DN cells were generated and examined by western blot as in (A). (E) Cells in (D) were treated as in (B) and immunostained for p-p38. All bars, 10 μm. (F) Cellular quiescence in C4-2b-Vector or C4-2b-p38DN cells after TGFβ2 or GDF10 incubation was determined as in (C). n cells were followed in multiple experiments: C4-2b-Vector (N=4–5), C4-2b-p38DN (N=3–5).
Figure Legend Snippet: Dominant-negative p38MAPK prevents dormancy induction by TGFβ2 or GDF10 in PCa cells in vitro (A) C4-2B4 cells stably-expressing empty vector or p38DN were examined by western blot for p-p38 followed by p38MAPK. β-tubulin, loading control. (B) Cells in (A) were treated with 50 ng/ml TGFβ2 or GDF10 for 60 min and immunostained for p-p38. (C) Cells in (A) were treated with TGFβ2 or GDF10 for 72 h. Cellular quiescence was determined by live-cell imaging. n cells were monitored in multiple experiments: C4-2B4-Vector (N=5–7), C4-2B4-p38DN (N=4–5). P values are by t test. (D) C4-2b-Vector or C4-2b-p38DN cells were generated and examined by western blot as in (A). (E) Cells in (D) were treated as in (B) and immunostained for p-p38. All bars, 10 μm. (F) Cellular quiescence in C4-2b-Vector or C4-2b-p38DN cells after TGFβ2 or GDF10 incubation was determined as in (C). n cells were followed in multiple experiments: C4-2b-Vector (N=4–5), C4-2b-p38DN (N=3–5).

Techniques Used: Dominant Negative Mutation, In Vitro, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Live Cell Imaging, Generated, Incubation

Related Articles

Western Blot:

Article Title: Dual-Specificity Phosphatase 4 Regulates STAT5 Protein Stability and Helper T Cell Polarization*
Article Snippet: .. Western blotting Antibodies against STAT5 (Cell Signaling), phospho-STAT5 (Tyr-694) (Cell Signaling), and β-tubulin (GeneTex) were purchased. .. Polyclonal rabbit antibodies against DUSP4 were generated by peptide immunization (YDERSPRAESLREDSTV-C).

Purification:

Article Title: Keratinocyte differentiation induces APOBEC3A, 3B, and mitochondrial DNA hypermutation
Article Snippet: .. Anti-HSP-60 (GeneTex, Irvine, California, GTX110089) and β-tubulin (GeneTex, GTX101279) antibodies were used to validate the purification of the mitochondrial and cytosolic fractions, respectively. .. Expression vectors and transfection pmax GFP was obtained from Lonza (Basal, Switzerland), and pmax A3A was constructed by inserting an A3A open reading frame (NM_145699) with EcoRI/XhoI ends into an AgeI/XhoI arm of pmax GFP.

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    GeneTex α tubulin
    Effect of BBR on HDAC activity and acetylation of histone H3 and <t>α-tubulin.</t> (a) Total cells lysates were incubated with various doses of BBR or 5 μM SAHA in HDAC assay buffer for 3 h. HDAC activity was initiated by adding the HDAC substrate and incubating at 37°C for 1 h. HDAC activity was measured by detecting the OD value at 405 nm. (b) MDA-MB-231 cells were treated with 25 or 50 μM BBR for 24 ~ 72 h or 1 μM SAHA for 24 h. Protein expressions of Ac-histone H3, Ac-α-tubulin and β-actin were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot. (c) MDA-MB-231 cells were treated with 50 μM BBR for 48 h, and nuclear and cytosolic extracts were prepared as described in “Methods”. Protein expressions of Ac-histone H3, histone H3, Ac-α-tubulin, α-tubulin, HDAC1, HDAC2, HDAC6, and GAPDH were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot.
    α Tubulin, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GeneTex rabbit anti α tubulin
    NNIMA (never in mitosis gene a)-related kinase 1 (NEK1) localization and protein levels are disrupted in the absence of senataxin. ( a ) Immunoblotting from testes extracts revealed a decrease in NEK1 protein levels in Setx −/− as compared with Setx +/+ spermatocytes. <t>α-Tubulin</t> was used as a loading control. Quantitation of NEK1 band intensity staining (shown as fold change) in Setx +/+ and Setx −/− . ( b ) NEK1 was observed to localize specifically to the axial element of the autosomes with a stronger intensity on the XY chromosomes in Setx +/+ spermatocytes, whereas NEK1 in Setx −/− spermatocytes was not associated with the XY chromosomes but was scattered throughout the nucleus. Quantitation of NEK1 fluorescence intensity staining (shown as fold change) in Setx +/+ and Setx −/− . Scale bar, 20 μm. Dotted circle, XY chromosomes. All data were plotted as the mean±s.d. Statistical analysis was performed using the Student’s t -test, * P
    Rabbit Anti α Tubulin, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of BBR on HDAC activity and acetylation of histone H3 and α-tubulin. (a) Total cells lysates were incubated with various doses of BBR or 5 μM SAHA in HDAC assay buffer for 3 h. HDAC activity was initiated by adding the HDAC substrate and incubating at 37°C for 1 h. HDAC activity was measured by detecting the OD value at 405 nm. (b) MDA-MB-231 cells were treated with 25 or 50 μM BBR for 24 ~ 72 h or 1 μM SAHA for 24 h. Protein expressions of Ac-histone H3, Ac-α-tubulin and β-actin were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot. (c) MDA-MB-231 cells were treated with 50 μM BBR for 48 h, and nuclear and cytosolic extracts were prepared as described in “Methods”. Protein expressions of Ac-histone H3, histone H3, Ac-α-tubulin, α-tubulin, HDAC1, HDAC2, HDAC6, and GAPDH were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot.

    Journal: Scientific Reports

    Article Title: A gene expression signature-based approach reveals the mechanisms of action of the Chinese herbal medicine berberine

    doi: 10.1038/srep06394

    Figure Lengend Snippet: Effect of BBR on HDAC activity and acetylation of histone H3 and α-tubulin. (a) Total cells lysates were incubated with various doses of BBR or 5 μM SAHA in HDAC assay buffer for 3 h. HDAC activity was initiated by adding the HDAC substrate and incubating at 37°C for 1 h. HDAC activity was measured by detecting the OD value at 405 nm. (b) MDA-MB-231 cells were treated with 25 or 50 μM BBR for 24 ~ 72 h or 1 μM SAHA for 24 h. Protein expressions of Ac-histone H3, Ac-α-tubulin and β-actin were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot. (c) MDA-MB-231 cells were treated with 50 μM BBR for 48 h, and nuclear and cytosolic extracts were prepared as described in “Methods”. Protein expressions of Ac-histone H3, histone H3, Ac-α-tubulin, α-tubulin, HDAC1, HDAC2, HDAC6, and GAPDH were analyzed by Western blotting. Images of each indicated probe were cropped from the same blot.

    Article Snippet: Antibodies specific for β-actin (GTX109639; 1:15000), AKT1 (GTX110613; 1:7500), phospho-Thr308-AKT1 (GTX61798; 1:500), AMPKα1 (GTX112998; 1:5000), phospho-Thr172-AMPKα (GTX63165; 1:2000), ATG5 (GTX113309; 1:2500), eIF2α (GTX101241; 1:250), phospho-Ser51-eIF2α (GTX50300; 1:1500), GAPDH (GTX100118; 1:30000), HDAC1 (GTX100513; 1:5000), HDAC2 (GTX109642; 1:5000), HDAC6 (GTX100722; 1:10000), histone H3 (GTX122148; 1:1000), acetyl-histone H3 (GTX122648; 1:5000), LC3B (GTX127375; 1:10000), mTOR (GTX101558; 1:500), phospho-Ser2448-mTOR (GTX62472; 1:2000), p70S6K (GTX103174; 1:500), phospho-p70S6K (GTX62758; 1:1500), and α-tubulin (GTX112141; 1:5000) were purchased from GeneTex.

    Techniques: Activity Assay, Incubation, Histone Deacetylase Assay, Multiple Displacement Amplification, Western Blot

    Virion encapsidation and restriction by A3G wild type and mutants. a , b Immunoblotting with FLAG antibody was used to detect transfected A3G wild type and mutants expressed in 293T virus producer cells and encapsidated into VSV-G pseudotyped virions in the absence, ΔVif (A) or presence, +Vif (B) of the A3 antagonist, Vif. The cell lysate and virion loading controls were α-tubulin and p24, respectively. The relative A3G levels shown below blots were calculated by setting the A3G wild type condition to 1 and determining the relative values of other lanes. One representative blot from three independent experiments is shown. c Infectivity in the absence or presence of Vif was measured by β-galactosidase activity in TZM-bl reporter cells. Results were normalized to the no A3 condition. Error bars represent the standard deviation of the mean calculated from three independent experiments. d The relative amount of proviral DNA integration in infected 293T cells in the presence of A3G wild type and mutants in comparison to the No A3 condition was determined by qPCR. Error bars represent the standard deviation of the mean calculated from at least two independent experiments. e The result of deaminase activity assay in lysates from 293T cells expressing hA3G and mutants. Error bars represent the standard deviations of the mean calculated from three independent experiments. Source data are provided in the Source Data file.

    Journal: Nature Communications

    Article Title: Understanding the structural basis of HIV-1 restriction by the full length double-domain APOBEC3G

    doi: 10.1038/s41467-020-14377-y

    Figure Lengend Snippet: Virion encapsidation and restriction by A3G wild type and mutants. a , b Immunoblotting with FLAG antibody was used to detect transfected A3G wild type and mutants expressed in 293T virus producer cells and encapsidated into VSV-G pseudotyped virions in the absence, ΔVif (A) or presence, +Vif (B) of the A3 antagonist, Vif. The cell lysate and virion loading controls were α-tubulin and p24, respectively. The relative A3G levels shown below blots were calculated by setting the A3G wild type condition to 1 and determining the relative values of other lanes. One representative blot from three independent experiments is shown. c Infectivity in the absence or presence of Vif was measured by β-galactosidase activity in TZM-bl reporter cells. Results were normalized to the no A3 condition. Error bars represent the standard deviation of the mean calculated from three independent experiments. d The relative amount of proviral DNA integration in infected 293T cells in the presence of A3G wild type and mutants in comparison to the No A3 condition was determined by qPCR. Error bars represent the standard deviation of the mean calculated from at least two independent experiments. e The result of deaminase activity assay in lysates from 293T cells expressing hA3G and mutants. Error bars represent the standard deviations of the mean calculated from three independent experiments. Source data are provided in the Source Data file.

    Article Snippet: The lysates were then subjected to Western blot with anti-FLAG M2 mAb from mouse (Cat # F3165, Sigma, 1:3,000), anti-α-tubulin mAb from mouse (Cat # GT114, GeneTex, 1:5,000) and anti-Vif mAb from mouse (NIH AIDS Reagent Program #319, 1:2,000) as primary antibodies.

    Techniques: Transfection, Infection, Activity Assay, Standard Deviation, Real-time Polymerase Chain Reaction, Expressing

    MPT0G211 sensitized MOLT-4 cells to vincristine-mediated mitotic arrest. a Cell cycle distributions of cells exposed to MPT0G211, tubastatin A (TBA), vincristine (VCR), or the indicated combination therapy for 24 h. A statistical analysis of the proportions of cells in the G2/M phase is shown in the right panel. b The levels of the apoptotic proteins caspase 3 and poly-ADP ribose polymerase (PARP) and the pro-survival proteins BCL-XL, BCL-2, and survivin were determined in cells treated with MPT0G211 (3 μM) or TBA (3 μM) in combination with VCR (1 nM) for 24 h. c The protein levels of cyclin B1, aurora B, p-CDC2, p-PLK, p-Histone 3, and MPM2 were evaluated following treatment with a combination of MPT0G211 or TBA with VCR for 24 h. d Cells were co-treated with MPT0G211 or TBA with VCR for 24 h and incubated with an α-tubulin antibody or DAPI. Microtubule dynamics were evaluated using a ZEISS LS 510META confocal microscope (magnification × 630). Scale bar = 20 μM. e Antitumor activity of MPT0G211 plus vincristine in a MOLT-4 xenograft model. When the tumor size reached 200 mm 3 , mice were injected with vehicle, vincristine (1 mg/kg, i.p., qwk), and MPT0G211 (30 mg/kg, i.p., qd) alone or a combination of both. The curves of tumor growth volume were expressed as mean ± SEM. f Changes of body weight after treatment. Data are shown as means ± standard errors of the means. * p

    Journal: Clinical Epigenetics

    Article Title: The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

    doi: 10.1186/s13148-018-0595-8

    Figure Lengend Snippet: MPT0G211 sensitized MOLT-4 cells to vincristine-mediated mitotic arrest. a Cell cycle distributions of cells exposed to MPT0G211, tubastatin A (TBA), vincristine (VCR), or the indicated combination therapy for 24 h. A statistical analysis of the proportions of cells in the G2/M phase is shown in the right panel. b The levels of the apoptotic proteins caspase 3 and poly-ADP ribose polymerase (PARP) and the pro-survival proteins BCL-XL, BCL-2, and survivin were determined in cells treated with MPT0G211 (3 μM) or TBA (3 μM) in combination with VCR (1 nM) for 24 h. c The protein levels of cyclin B1, aurora B, p-CDC2, p-PLK, p-Histone 3, and MPM2 were evaluated following treatment with a combination of MPT0G211 or TBA with VCR for 24 h. d Cells were co-treated with MPT0G211 or TBA with VCR for 24 h and incubated with an α-tubulin antibody or DAPI. Microtubule dynamics were evaluated using a ZEISS LS 510META confocal microscope (magnification × 630). Scale bar = 20 μM. e Antitumor activity of MPT0G211 plus vincristine in a MOLT-4 xenograft model. When the tumor size reached 200 mm 3 , mice were injected with vehicle, vincristine (1 mg/kg, i.p., qwk), and MPT0G211 (30 mg/kg, i.p., qd) alone or a combination of both. The curves of tumor growth volume were expressed as mean ± SEM. f Changes of body weight after treatment. Data are shown as means ± standard errors of the means. * p

    Article Snippet: Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA).

    Techniques: Incubation, Microscopy, Activity Assay, Mouse Assay, Injection

    The effects of MPT0G211 on HL-60 and MOLT-4 cell growth and cell cycle progression. a Acetyl-α-tubulin and acetyl-histone 3 were detected in cells treated with MPT0G211 or tubastatin A (TBA) for 24 h. b HL-60, MOLT-4, and HUVECs were incubated with different concentrations of MPT0G211 or TBA for 48 h. Cell viability was evaluated using an MTT assay. c HL-60 and d MOLT-4 cells were treated with MPT0G211 or TBA for 48 h and analyzed by flow cytometry to assess cell cycle distribution. Data are shown as means ± standard errors of the means. * p

    Journal: Clinical Epigenetics

    Article Title: The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

    doi: 10.1186/s13148-018-0595-8

    Figure Lengend Snippet: The effects of MPT0G211 on HL-60 and MOLT-4 cell growth and cell cycle progression. a Acetyl-α-tubulin and acetyl-histone 3 were detected in cells treated with MPT0G211 or tubastatin A (TBA) for 24 h. b HL-60, MOLT-4, and HUVECs were incubated with different concentrations of MPT0G211 or TBA for 48 h. Cell viability was evaluated using an MTT assay. c HL-60 and d MOLT-4 cells were treated with MPT0G211 or TBA for 48 h and analyzed by flow cytometry to assess cell cycle distribution. Data are shown as means ± standard errors of the means. * p

    Article Snippet: Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA).

    Techniques: Incubation, MTT Assay, Flow Cytometry, Cytometry

    Ectopic expression of HDAC6 rescues cells from apoptosis induced by MPT0G211/doxorubicin combination. a HDAC6 levels were measured 18 h after transfection with an HDAC6-expression plasmid. b Following co-treatment with MPT0G211 and doxorubicin (DOXO) for 24 h, the levels of the apoptotic proteins poly-ADP ribose polymerase (PARP), caspase 3, γ-H2AX, and acetyl-α-tubulin were measured in HDAC6-overexpressing cells. c Cell cycle distribution was measured in HDAC6-transfected HL-60 cells following treatment with a combination of MPT0G211 and DOXO. A quantitative analysis of the proportions of cells in the sub-G1 phase of cell cycle is shown in d . Data are shown as means ± standard errors of the means. * p

    Journal: Clinical Epigenetics

    Article Title: The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

    doi: 10.1186/s13148-018-0595-8

    Figure Lengend Snippet: Ectopic expression of HDAC6 rescues cells from apoptosis induced by MPT0G211/doxorubicin combination. a HDAC6 levels were measured 18 h after transfection with an HDAC6-expression plasmid. b Following co-treatment with MPT0G211 and doxorubicin (DOXO) for 24 h, the levels of the apoptotic proteins poly-ADP ribose polymerase (PARP), caspase 3, γ-H2AX, and acetyl-α-tubulin were measured in HDAC6-overexpressing cells. c Cell cycle distribution was measured in HDAC6-transfected HL-60 cells following treatment with a combination of MPT0G211 and DOXO. A quantitative analysis of the proportions of cells in the sub-G1 phase of cell cycle is shown in d . Data are shown as means ± standard errors of the means. * p

    Article Snippet: Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation

    NNIMA (never in mitosis gene a)-related kinase 1 (NEK1) localization and protein levels are disrupted in the absence of senataxin. ( a ) Immunoblotting from testes extracts revealed a decrease in NEK1 protein levels in Setx −/− as compared with Setx +/+ spermatocytes. α-Tubulin was used as a loading control. Quantitation of NEK1 band intensity staining (shown as fold change) in Setx +/+ and Setx −/− . ( b ) NEK1 was observed to localize specifically to the axial element of the autosomes with a stronger intensity on the XY chromosomes in Setx +/+ spermatocytes, whereas NEK1 in Setx −/− spermatocytes was not associated with the XY chromosomes but was scattered throughout the nucleus. Quantitation of NEK1 fluorescence intensity staining (shown as fold change) in Setx +/+ and Setx −/− . Scale bar, 20 μm. Dotted circle, XY chromosomes. All data were plotted as the mean±s.d. Statistical analysis was performed using the Student’s t -test, * P

    Journal: Cell Discovery

    Article Title: Senataxin controls meiotic silencing through ATR activation and chromatin remodeling

    doi: 10.1038/celldisc.2015.25

    Figure Lengend Snippet: NNIMA (never in mitosis gene a)-related kinase 1 (NEK1) localization and protein levels are disrupted in the absence of senataxin. ( a ) Immunoblotting from testes extracts revealed a decrease in NEK1 protein levels in Setx −/− as compared with Setx +/+ spermatocytes. α-Tubulin was used as a loading control. Quantitation of NEK1 band intensity staining (shown as fold change) in Setx +/+ and Setx −/− . ( b ) NEK1 was observed to localize specifically to the axial element of the autosomes with a stronger intensity on the XY chromosomes in Setx +/+ spermatocytes, whereas NEK1 in Setx −/− spermatocytes was not associated with the XY chromosomes but was scattered throughout the nucleus. Quantitation of NEK1 fluorescence intensity staining (shown as fold change) in Setx +/+ and Setx −/− . Scale bar, 20 μm. Dotted circle, XY chromosomes. All data were plotted as the mean±s.d. Statistical analysis was performed using the Student’s t -test, * P

    Article Snippet: Membranes were the blocked in 5% skim milk in TBS/Tween-20 or 5% BSA/TBS/Tween-20 for 1 h and immunoblotting was performed with the following primary antibodies diluted 1:1 000 in 5% skim milk/TBS/Tween-20 or 5% BSA/TBS/Tween-20 at 4 °C overnight: goat anti-ATR (Santa Cruz Biotechnology; SC-1887), rabbit anti-NEK1 (Bioss Inc., BS-7814R), rabbit anti-ATRIP (Thermo Scientific; PA1-519), mouse monoclonal anti-CHK1 (Cell Signaling Technology; 2 360), rabbit anti-CHD4 (Abcam; AB72418), sheep anti-Setx (in-house), rabbit anti-TopBP1 (Abcam; AB2402), rabbit anti SUMO-1 (Abcam; AB32058), rabbit anti-phospho ATR (S428) (Cell Signaling Technology; 2 853), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (Genetex; GTX100118) and rabbit anti-α tubulin (Genetex; GTX 112141).

    Techniques: Quantitation Assay, Staining, Fluorescence